Sergienko Eduard A, Jordan Frank
Department of Chemistry and Program in Cellular and Molecular Biodynamics, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Biochemistry. 2002 Mar 26;41(12):3952-67. doi: 10.1021/bi011860a.
Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants suggested that the first transition corresponded to substrate binding in an active site and a subsequent step responsible for conversion to an asymmetric conformation. Consequently, the second enzyme state may report on "unregulated" enzyme, since the regulatory site does not participate in its generation. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substrate dependence of initial rate. The second transition corresponds to binding a substrate molecule in the regulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds to the allosterically regulated conformation, previously referred to as activated enzyme. The pH dependence of the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additional conformations of the enzyme.
酵母丙酮酸脱羧酶（YPDC，EC 4.1.1.1）在产物（乙醛）形成的进程曲线中表现出明显的延迟期。目前被广泛接受的YPDC动力学模型预测，只有当底物结合到调节位点时，一个缓慢的激活步骤才会将无活性的酶转化为活性形式。这种别构行为导致了S形的稳态动力学。YPDC的E477Q活性位点变体在低pH值下呈现双曲线型的初始速率曲线，这与该模型不一致。该变体产物形成的进程曲线呈S形，这与存在三种具有不同稳态速率的相互转化构象相一致。令人惊讶的是，pH≤5.0时的野生型YPDC也具有S形的进程曲线，对应中间稳态的构象是最活跃的。在4.5 - 6.5的pH范围内对野生型YPDC的底物激活进行重新研究发现，在所有pH值下都有两个特征性转变。稳态速率的值是pH和底物浓度的函数，影响进程曲线是呈现“正常”形状还是带有拐点的S形。表观速率常数对底物的依赖性表明，第一个转变对应底物在活性位点的结合以及随后负责转化为不对称构象的步骤。因此，第二种酶状态可能代表“未受调节”的酶，因为调节位点不参与其形成。这种酶状态利用交替位点机制，导致初始速率对底物的双曲线依赖性。第二个转变对应底物分子在调节位点的结合以及随后的微小构象调整。第三种酶状态对应别构调节的构象，之前被称为激活酶。希尔系数的pH依赖性表明丙酮酸在野生型YPDC的调节位点和活性位点随机结合。向YPDC中添加丙酮酰胺或乙醛会导致酶出现额外的构象。
